This invention is in the area of synthetic organic chemistry, and is in particular carboranyl containing synthetic nucleosides and oligonucleotides, and their method of preparation and use.
The goal of cancer therapy is to achieve a degree of selectivity that spares normal cells and destroys all malignant ones, since even a small number of remaining malignant cells can lead to recurrence, metastasis, and death. A two-component or binary system comprised of constituents that alone are nonlethal and largely confined to malignant cells, and which when combined are lethal to the neoplastic cells yet innocuous to normal cells is an ideal modality. One advantage of this type of binary system is that each component can be manipulated independently to maximize selectivity.
Boron neutron capture therapy (BNCT, see FIG. 1) is a binary system which combines two separately nonlethal constituents, a radiosensitizing compound that contains a stable boron-10(10B) isotope, and nonionizing neutron radiation. When boron-10 is irradiated with neutrons, a nuclear reaction occurs that yields helium nuclei (xcex1-particle), lithium nuclei, and about 100 million times more energy than the initial irradiated energy. The generated radiation destroys malignant cells containing the boron compound. Selectivity is achieved through the use of compounds which accumulate primarily in malignant cells and/or by aiming the neutron beam at the tumor mass which contains the boron carrier.
The major obstacles in BNCT are: (1) the achievement of a sufficiently high intracellular boron concentration and (2) selectivity toward tumor cells. Although attempts to develop tumor-selective boron compounds date back to the 1960s and despite extensive studies, the problem of selective delivery of boron carriers to tumor cells remains.
Many classes of compounds have been synthesized for BNCT. For example, see Barth, R. F.; Soloway, A. H.; Fairchild, R. G.; Brugger, R. M. Cancer 1992, 70, 2995-3008; Fairchild, R. G.; Kahl, S. B.; Laster, B. H.; Kalef-Ezra, J.; Popenoe, E. A. Cancer Res. 1990, 50, 4860-4865; and Zamenhof, R. G.; Kalend, A. M.; and Bloomer, W. D. J Natl Cancer Inst 1992, 84, 1290-1291.
The first boron-containing nucleoside, 5-dihydroxyboryl-2xe2x80x2-deoxyuridine, was synthesized by Schinazi and Prusoff in 1978. Schinazi, R. F., Prusoff, W. H. Tetrahedron Lett 1978, 4981-4984; and Schinazi, R. F.; Prusoff, W. H. J Orgr Chem 1985, 50, 841-847. Sood et al. have reported the synthesis of a series of cyanoborane adducts of 2xe2x80x2-deoxynucleosides, specifically 2xe2x80x2-deoxyguanosine-N7-cyanoborane, 2xe2x80x2-deoxyinosine-N7-cyanoborane, 2xe2x80x2-deoxyadenosine-N1-cyanoborane, and-2xe2x80x2-deoxycytidine-N3-cyanoborane. Sood, A.; Spielvogel, B. F.; Shaw, B. R. J Am Chem Soc 1989, 111, 9234-9235.
Sood et al. have also reported the synthesis of oligonucleotides with a boronated internucleotide backbone, in the form of boranophosphates and boranophosphate methyl esters. The borane (BH3) group in these boronated oligonucleotides is isoelectronic and isostructural with normal O-oligonucleotides and oligonucleotide methylphosphonates. Sood, A.; Shaw, B. R.; Spielvogel, B. F. J Am Chem Soc 1990, 112, 9000-9001. The Sood compounds in general have a low boron content and some have lower than desired lipophilicity.
U.S. Pat. No. 5,130,302 to Spielvogel, et al., discloses a novel class of boronated nucleosides, nucleotides and oligonucleotides for use as antineoplastic, antiinflammatory, and antihypertensive agents. The nucleosides, nucleotides and oligonucleotides are covalently attached to either BH2CN, BH3, or BH2CO2R moieties, wherein R is C1 to C18 alkyl.
A number of carboranyl pyrimidines have been prepared for use in boron neutron capture therapy. Examples of carboranyl pyrimidines include 5-(3-o-carboranylpropyl-6-methyl-2-thiouracil (compound A) (Wilson, J. G. Pigment Cell Res 1989, 2, 297-303), 2,4-dichloro-5-(1-o-carboranylmethyl)-6-methylpyrimidine; (compound B) (Reynolds, R. C.; Trask, T. W.; Sedwick, W. D. J Org Chem 1991, 56, 2391-2395); and 5-carboranyluracil (compound C) (Goudgaon, N. M.; El-Kattan, Y.; Fulcrand, G.; Liotta, D. C.; Schinazi, R. F. IMEBORON VIII, Knoxyille, Tenn.; p72, 1993).
Purine and pyrimidine nucleosides that contain a carboranyl group attached to the purine or pyrimidine base have also been reported. Yamamoto, Y.; Seko, T.; Nakamura, H. Heteroatom Chem 1992, 3, 239-244; and Schinazi, R. F.; Goudgaon, N. M.; Soria, J.; Liotta, D. C. 5th International Symposium on Neutron Capture Therapy, Columbus, Ohio; p11, 1992; Schinazi, R. F.; Goudgaon, N.; Soria, J.; Liotta, D. C. Tenth International Roundtable: Nucleosides and Nucleotides, Park City, Utah; p28, 1992. These compounds are lipophilic and some are readily phosphorylated by cellular kinases, and in certain cells can incorporate into DNA as analogues of natural 2xe2x80x2-deoxypyrimidine nucleosides. Examples include 5-carboranyl-2xe2x80x2-deoxyuridine (compound D, CDU), 5-carboranyluridine (compound E, CU), 5-(1-hydroxymethyl)carboranyluridine, and 5-(1-hydroxymethyl)carboranyluridine (compound F, HMCU). 
PCT WO 93/17028 filed by Raymond F. Schinazi and Dennis C. Liotta discloses a number of synthetic nucleosides that contain a carboranyl moiety covalently attached to a purine or pyrimidine base, wherein the sugar moiety optionally contains a second heteroatom in the 3xe2x80x2-position of the ring. Preferred compounds are 2-hydroxymethyl-5-(5-carboranylcytosin-1-yl)-1,3-oxathiolane (compound G) and 2-hydroxymethyl-5-(5-carboranyluridin-1-yl)-1,3-oxathiolane (compound H).
Powell, et al., recently reported the synthesis of oligonucleotides that contain 3xe2x80x2,5xe2x80x2-nido-o-carboranyl-phosphoramidate linkages (compound I). While the oligonucleotide could reportedly localize in the cell nucleus, the boron moiety is acid labile because it is linked to the phosphorus atom through an amide-type bond.
The requirements for efficient BNCT with oligonucleotides, which include cell selectivity (ability to accumulate preferentially in diseased cells), stability of the chemotherapeutic agent in vivo (resistance against digestion by cellular nucleases and chemical stability), and transportability. (ability of the chemotherapeutic agent to pass easily through cellular membranes), are very similar to the requirements for Antisense oligonucleotide Technology (AOT), another recently developed therapy for cancer as well as other diseases. Uhlmann, xe2x80x9cAntisense Oligonucleotides: A New Therapeutic Approachxe2x80x9d Chemical Reviews, 90(4), June 1990. The compounds should also be relatively non-toxic. Antisense technology refers in general to the modulation of gene expression through a process wherein a synthetic oligonucleotide is hybridized to a complementary nucleic acid sequence to inhibit transcription or replication (if the target sequence is DNA), inhibit translation (if the target sequence is RNA) or to inhibit processing (if the target sequence is pre-RNA). A wide variety of cellular activities can be modulated using this technique. A simple example is the inhibition of protein biosynthesis by an antisense oligonucleotide bound to mRNA. In another embodiment, a synthetic oligonucleotide is hybridized to a specific gene sequence in double stranded DNA, forming a triple stranded complex (triplex) that inhibits the expression of that gene sequence. Antisense oligonucleotides can be also used to activate gene expression indirectly by suppressing the biosynthesis of a natural repressor or directly by reducing termination of transcription. AOT can be used to inhibit the expression of pathogenic genes, for example, those that facilitate the replication of viruses, including human immunodeficiency virus (HIV), hepatitis B virus (HBV), and herpesviruses, and cancers, particularly solid tumor masses such as gliomas, breast cancer, and melanomas.
While progress has been made in the areas of both BNCT and AOT, none of the synthetic oligonucleotides prepared to date exhibit the optimal combination of cell selectivity, stability in vivo, and ability to pass easily through cellular membranes (transportability).
Therefore, it is an object of the present invention to provide a new class of synthetic oligonucleotides for use in BCNT, AOT, or both, that exhibit a desired profile of cell selectivity, stability in vivo, and ability to pass easily through cellular membranes.
It is another object of the present invention to provide new methods for the preparation of boron-containing nucleosides and oligonucleotides.
Carboranyl-containing nucleosides and oligonucleotides are provided for use in boron neutron capture therapy (BNCT) that are lipophilic and have a high content of boron atoms. In one embodiment, dinucleotides and oligonucleotides are provided that contain at least one uncharged 3xe2x80x2,5xe2x80x2-O,O-[(carboran-1-yl-methyl)phosphonate] internucleotide linkage in place of the naturally occurring 3xe2x80x2,5xe2x80x2-O,O-phosphodiester residue. The (carboran-1-yl-methyl)phosphonate linkage is not degraded by nucleases, and therefore, dinucleotides and oligonucleotides that contain the 3xe2x80x2,5xe2x80x2-O,O-[(carboran-1-yl-methyl)phosphonate] internucleotide linkage are stable in biological fluids and cells. In light of the fact that oligonucleotides are primarily degraded by 3xe2x80x2-exonucleases, in a preferred embodiment, oligonucleotides are provided in which the two terminal nucleosides at the 3xe2x80x2-end, or nucleosides adjacent to these nucleosides, are linked via the nuclease-stable 3xe2x80x2,5xe2x80x2-O,O-[(o-carboran-1-yl-methyl)phosphonate] bridge. The 3xe2x80x2,5xe2x80x2-O,O-[(o-carboran-1-yl-methyl)phosphonate] bridge is also stable in acidic environments and is highly thermally stable.
Oligonucleotides can be designed for BNCT according to methods described herein that are complementary to overexpressed or unique RNA or DNA sequences in target cancer cells, as a means to selectively accumulate the boron-containing material into these cells. Oligonucleotides of specific gene sequences that include one or more 3xe2x80x2,5xe2x80x2-linking-(carboran-1-yl)phosphonate moieties can also be used in antisense therapy in the selective modification of gene expression.
In a second embodiment, nucleosides are provided that bear a (carboran-1-yl)phosphonate moiety in the 3xe2x80x2 and/or 5xe2x80x2-position. These synthetic nucleotides are useful in boron neutron capture therapy, and selected compounds can exhibit activity against viruses such as HIV and HBV.
In another embodiment, oligonucleotides are provided that bear a carboranyl-modified base in at least one of the nucleosides of the oligomer. In a preferred embodiment, the carboranyl-containing base is in a nucleoside located at the 3xe2x80x2-terminus, in the nucleoside adjacent to the 3xe2x80x2-terminal nucleoside, in the 5xe2x80x2-terminal nucleoside, or in the nucleoside adjacent to the 5xe2x80x2-terminal nucleoside. oligonucleotides bearing carboranyl-containing bases in the 3xe2x80x2-terminal nucleoside or the nucleoside adjacent to the 3xe2x80x2-terminal are more resistant to degradation by 3xe2x80x2-exonucleases. It has been discovered that olignucleotides bearing carboranyl-containing base units in the preferred positions hybridize more effectively to complementary nucleic acid sequences than oligonucleotides bearing carboranyl-containing bases in other positions.
In yet another embodiment, oligonucleotides are provided that bear at least one 3xe2x80x2,5xe2x80x2-[(O,O-carboran-1-yl) phosphonate] residue and at least one nucleoside that contains a carboranyl-containing base, as a means to increase the boron density and lipophilicity of the molecule, and depending on the location of the modifications, increase the stability of the oligomer in vivo in biological fluids or cells.
In another embodiment of the invention, nucleosides and oligonucleotides bearing an xe2x80x94O-[(carboran-1-yl)alkyl]phosphate, S-[(carboran-1-yl)alkyl]phosphorothioate, or Se-[(carboran-1-yl)alkyl]phosphoroselenoate in place of the (carboran-1-yl)phosphonate moiety are provided.
In addition to the use of the oligonucleotides described herein in BNCT, the oligomers disclosed herein can be used in vitro to carry out structure-activity relationships on the bulk tolerance of hybridization of synthetic oligonucleotides with complementary nucleic acid sequences, in MRI imaging, or as probes in a variety of diagnostic techniques.
Carboranyl-containing oligonucleotides can also be used to effect mutation of expressed HIV-1 reverse transcriptase, using in vitro or in vivo Site Directed Mutagenesis (SDM).
Nucleosides, nucleotides, and oligonucleotides can be prepared that contain boron clusters as a means to enhance lipophilicity wherein the boron is not enriched in 10B, but instead, in the 11B isotope. The nucleosides, nucleotides, and oligonucleotides of the present invention that are used for BNCT or other diagnostic techniques that depend on neutron radiation decay for the destruction of diseased cells or for signaling purposes should be enriched with a suitable amount of 10B, normally approximately 90-100% 10B, and typically between 92-96% 10B.
A novel process is provided for the preparation of nucleosides, dinucleotides, and oligonucleotides containing an (carboran-1-yl-methyl)phosphonate moiety via the key starting material O-methyl(carboran-1-yl)methyl phosphonate.